The red cell membrane has proven to be an accessible model from which most basic understanding of membrane architecture has been derived. Despite extensive study, the red cell membrane remains a rich source of still unidentified proteins, and analysis of these new proteins will provide molecular insight into human hemolytic diseases. Three specific aims are proposed which should significantly extend the limits of our knowledge of membrane biology. I. Rh antigens. Despite major clinical importance, the molecular understanding of the Rh antigens remains primitive. The Rh antigens are known to contain at least two subunits, the Mr 32kDa Rh-acylproteins and the Mr 40-60 kDa Rh-glycoproteins. Molecular studies will be undertaken to structurally define protein polymorphisms and posttranslational modifications of the Rh proteins and the organization of the Rh membrane complex. The cDNAs for the mouse Rh homologs will be isolated and used to study Rh expression and distribution in the mouse and during murine development. Human linkage studies will be performed to establish the genetic background for Rh antigenic variants. II. Palmitylated 55kDa membrane protein (p55). p55 is a recently identified peripheral red cell membrane protein with homology to src tyrosine kinase, yeast guanylate kinase, and a Drosophila tumor suppressor. p55 undergoes extremely rapid and reversible palmitylation which appears linked to membrane phospholipid metabolism. The abundance of p55 will be assessed in red cell membranes from human patients and certain mammalian species. The structure, organization, and fatty acylation of p55 will be studied biochemically. Identification of the function of p55 will be sought by comparative and site-directed mutagenesis studies. The tissue and developmental expression of p55 will be studied in mouse. III. Kindreds with hemolytic anemia. Two newly identified large kindreds with typical dominantly inherited spherocytosis will be studied for restriction fragment polymorphisms linked to red cell membrane proteins. Protein studies will be performed on the red cells and tissue sections from patients with neuroacanthocytosis and recessive spherocytosis. Blood samples will also be provided to collaborating investigators performing physical analyses.